Lipid-free Antigen B subunits from echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties

Background: The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied.<p></p> Methodology/Principal Findings: Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles.<p></p> Conclusions/Significance: We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.<p></p&gt

Structural Characterization and in vitro Lipid Binding Studies of Non-specific Lipid Transfer Protein 1 (nsLTP1) from Fennel (Foeniculum vulgare) Seeds

Non-specific lipid transfer proteins (nsLTPs) are cationic proteins involved in intracellular lipid shuttling in growth and reproduction, as well as in defense against pathogenic microbes. Even though the primary and spatial structures of some nsLTPs from different plants indicate their similar features, they exhibit distinct lipid-binding specificities signifying their various biological roles that dictate further structural study. The present study determined the complete amino acid sequence, in silico 3D structure modeling, and the antiproliferative activity of nsLTP1 from fennel (Foeniculum vulgare) seeds. Fennel is a member of the family Umbelliferae (Apiaceae) native to southern Europe and the Mediterranean region. It is used as a spice medicine and fresh vegetable. Fennel nsLTP1 was purified using the combination of gel filtration and reverse-phase high-performance liquid chromatography (RP-HPLC). Its homogeneity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The purified nsLTP1 was treated with 4-vinyl pyridine, and the modified protein was then digested with trypsin. The complete amino acid sequence of nsLTP1 established by intact protein sequence up to 28 residues, overlapping tryptic peptides, and cyanogen bromide (CNBr) peptides. Hence, it is confirmed that fennel nsLTP1 is a 9433 Da single polypeptide chain consisting of 91 amino acids with eight conserved cysteines. Moreover, the 3D structure is predicted to have four α-helices interlinked by three loops and a long C-terminal tail. The lipid-binding property of fennel nsLTP1 is examined in vitro using fluorescent 2-p-toluidinonaphthalene-6-sulfonate (TNS) and validated using a molecular docking study with AutoDock Vina. Both of the binding studies confirmed the order of binding efficiency among the four studied fatty acids linoleic acid \u3e linolenic acid \u3e Stearic acid \u3e Palmitic acid. A preliminary screening of fennel nsLTP1 suppressed the growth of MCF-7 human breast cancer cells in a dose-dependent manner with an IC50 value of 6.98 µM after 48 h treatment

Drug Discovery Using Chemical Systems Biology: Identification of the Protein-Ligand Binding Network To Explain the Side Effects of CETP Inhibitors

Systematic identification of protein-drug interaction networks is crucial to correlate complex modes of drug action to clinical indications. We introduce a novel computational strategy to identify protein-ligand binding profiles on a genome-wide scale and apply it to elucidating the molecular mechanisms associated with the adverse drug effects of Cholesteryl Ester Transfer Protein (CETP) inhibitors. CETP inhibitors are a new class of preventive therapies for the treatment of cardiovascular disease. However, clinical studies indicated that one CETP inhibitor, Torcetrapib, has deadly off-target effects as a result of hypertension, and hence it has been withdrawn from phase III clinical trials. We have identified a panel of off-targets for Torcetrapib and other CETP inhibitors from the human structural genome and map those targets to biological pathways via the literature. The predicted protein-ligand network is consistent with experimental results from multiple sources and reveals that the side-effect of CETP inhibitors is modulated through the combinatorial control of multiple interconnected pathways. Given that combinatorial control is a common phenomenon observed in many biological processes, our findings suggest that adverse drug effects might be minimized by fine-tuning multiple off-target interactions using single or multiple therapies. This work extends the scope of chemogenomics approaches and exemplifies the role that systems biology has in the future of drug discovery

Lipidomics in Health and Diseases - Beyond the Analysis of Lipids

published_or_final_versio

Bacterial nanocellulose and long-chain fatty acids interaction: an in silico study

Chronic wounds are a big challenge in contemporary society, as they lead to a decrease in life-quality, amputations and even death. Infections and biofilm formation might occur with chronic wounds, due to the higher susceptibility to antibiotic multi-resistant bacteria. In this situation, novel wound dressing biomaterials are needed for treatment. Thus, the aim of this research was to evaluate a possible BNC interaction with tucumã oil/butter-derived fatty acids, as this system could be a promising biomaterial for wound treating. The interaction between  cellobiose (BNC basic unit) and four fatty acids was evaluated by ab initio simulations and density functional theory (DFT), through SIESTA code. Molecular docking was also used to investigate the effect of a possible releasing of the studied fatty acids to the quorum-sensing proteins of Pseudomonas aeruginosa (gram-negative bacterium) and Staphylococcus aureus (gram-positive bacterium). According to ab initio simulations, the interaction between cellobiose and fatty acids derived from tucumã oil/butter was suggested due to physical adsorption (energy around 0.17-1.33 eV) of the lipidic structures into cellobiose. A great binding affinity (∆G ranging from 4.2-8.2 kcal.mol-1) was observed for both protonated and deprotonated fatty acids against P. aeruginosa (LasI, LasA and Rhlr) and S. aureus (ArgA and ArgC) quorum-sensing proteins, indicating that these bioactive compounds might act as potential antimicrobial and/or antibiofilm agents in the proposed system. Hence, from a theoretical viewpoint, the proposed system could be a promising raw biomaterial in the production of chronic wound dressings

Lipid-Free Antigen B Subunits from <i>Echinococcus granulosus</i>: Oligomerization, Ligand Binding, and Membrane Interaction Properties

The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied. Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles. We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid acquisition and/or transport between parasite tissues. These results may therefore indicate vulnerabilities open to targeting by new types of drugs for hydatidosis therapy.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Médica

Studies of cationic amphiphilic drug catalysed membrane degradation

Imperial Users onl

Effects of Synthetic Ligands on Heterodimer Pairs Regarding Full-Length Human PPARα, RXRα and LXRα

Nuclear receptor study is critically relevant in therapeutic medicine since the intricate details of disease states pertaining to atherosclerosis and diabetes are poorly understood. Three nuclear receptors of interest regulate target genes pertaining to cholesterol and fatty acid regulation, linking these receptors to therapeutic medicine. The first is the peroxisome proliferator-activated receptor alpha (PPARa), which resides in liver and muscle, coordinating lipoprotein and fatty acid homeostasis [1]. Cholesterol homeostasis is dictated by the liver X receptor alpha (LXRa), targeting genes pertaining to the kidney, intestine, liver and adipose tissues [2]. A common partner receptor to PPARa and LXRa is known as the retinoid X receptor alpha (RXRa) [3]. Although each receptor appears unique in function, the cause and effects of disease states are poorly understood due to the promiscuous nature of these receptor proteins. These particular receptors can form permissive heterodimers where metabolic effects can be manipulated by ligands [3]. Accordingly, clinical care becomes increasingly complex as synthetic ligands made to target one receptor could have additional repercussions. With respect to therapeutic medicine, ligand binding may not be exclusive. Therefore, it becomes necessary to study synthetic ligands with each receptor, individually and in heterodimeric form, to further understand the complex regulation and clinical implications of synthetic ligands on disease states such as atherosclerosis and diabetes

Probing Cancer Targets and Therapeutic Mechanisms using Small Molecules

Small molecules are a powerful tool to illuminate biological mechanisms and assist in the identification and validation of therapeutic targets. KRAS is the single most frequently mutated oncogene in human cancer, with particularly high mutation frequencies observed in pancreas (95%), colon (45%), and lung (35%) cancer. However, despite three decades of effort, there is no clinical viable KRAS cancer therapy. The first part of this thesis focuses on exploring the potential of directly targeting the KRAS nucleotide binding site. Directly targeting oncogenic KRAS with small molecules in the nucleotide-binding site has had limited success due to the high affinity of KRAS for nucleotide GTP and the high cellular concentration of GTP. The strategy of generating engineered KRAS allele based on shape and covalent complementarity was exploited herein to address this challenge. Using fragment-based small molecule design, a cell-membrane-permeable covalent inhibitor able to irreversibly modify the engineered nucleotide-binding site of KRAS was developed. The second part of this thesis describes the investigation of the therapeutic potential of imidazole ketone erastin (IKE), a small molecule inhibitor of the cystine/glutamate antiporter system xc–, in a subcutaneous xenograft model of Diffuse Large B Cell Lymphoma (DLBCL). A biodegradable polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticle formulation was employed to aid in the delivery of IKE to cancer cells in vivo. This IKE nanoparticle system showed improved tumor accumulation and therapeutic index relative to free IKE, indicating its potential for treating DLBCL. The final part of this thesis describes the study of lipid metabolism features of ferroptotic cell death using quantitative reverse transcription PCR (RT-qPCR) and mass spectrometry-based lipidomic analysis. In summary, this work illustrates how chemistry and chemical biology approaches can supplement existing efforts towards the design and discovery of new drugs for challenging targets, as well as aid in the study of therapeutic mechanisms

Description of Prototype Modes-of-Action Related to Repeated Dose Toxicity

This report presents the definition and detailed documentation of chosen toxicological MoAs associated with repeated dose target organ toxicity as a first step in building a "prototype" safety assessment framework. In addition to providing a detailed description of the two chosen MoAs related to chronic liver toxicity, namely "MoA from Protein Alkylation to Liver Fibrosis" and "MoA from Liver X Receptor Activation to Liver Steatosis", the report also describes the working process leading to this result including the problems that have been encountered. The exercise followed as far as possible relevant WHO-IPCS and OECD guidance. The report represents the first deliverable of a contract of work between Cosmetics Europe and the European Commission's Joint Research Centre for supplementing the work of the SEURAT-1 research cluster.JRC.I.5-Systems Toxicolog